6517761Vanderbilt Specialized Chemistry CenterMuscarinic Receptor 5 (M5) Positive Allosteric Modulator ACh foldshift Ca2013712Discovery of Novel Positive Allosteric Modulators (PAM) of the Muscarinic Receptor M5: Fold-shift AssayAssay Provider: P. Jeffrey ConnAssay Provider Affiliation: Vanderbilt UniversityMuscarinic acetylcholine receptors are family A GPCRs comprised of five distinct mammalian subtypes (mAChR1-5 or M1-M5), which are expressed differentially throughout the body and play an important role in a variety of physiological processes. Among the mAChRs, M1 and M4 have been historically considered attractive targets for small molecule treatments of numerous CNS disorders such as Alzheimer's disease and schizophrenia due to their respective localization and involvement in regulation of certain aspects of learning, memory, sleep, motor control, reward, and pain, among others. However, discovery of subtype-selective small molecules has proven highly difficult due to the conservation of the orthosteric binding-site across the mAChRs. This has contributed to the failure of muscarinic agonists in clinical trials and has also hampered pharmacological investigation into the role(s) of each mAChR in basic neurobiology.Among the mAChRs, M5 has remained perhaps the most challenging to investigate pharmacologically due in part to its extremely low expression level and a complete lack of M5-selective ligands. Interestingly, studies using M5-KO mice suggest that M5 is the sole mediator of acetylcholine-induced cerebrovasodilation, which has led to the hypothesis that an M5 activator would have therapeutic efficacy in treatment of cerebrovascular dementias and ischemic stroke. Furthermore, M5-KO mice show dramatically reduced reward responses to drugs of abuse, consistent with its putative localization on midbrain dopaminergic neurons of the nigrostriatal and mesolimbic pathways. This suggests that M5 antagonism or negative modulation may have utility in treatment of illicit drug addiction and withdrawal. Despite these and other related findings from M5-KO mice, there remains a strong need for small molecule tools to probe M5 function and test M5-related hypotheses in order to advance the state of the mAChR research field and provide critical proof-of-concept studies for drug discovery aims.Assay Info: CHO-K1 cells stably expressing human M5 were loaded with calcium indicator dye (2mM Fluo-4 AM) for 45-60 min at 37 degrees C. Dye was removed and replaced with the appropriate volume of assay buffer, pH 7.4 (1X HBSS (Hanks' Balanced Salt Solution), supplemented with 20 mM HEPES and 2.5 mM probenecid). All compounds were diluted in assay buffer for a 60 uM 2X stock concentration (30 uM final concentration) in 0.6% DMSO. This stock was then added to the assay plate for a final DMSO concentration of 0.3%. Acetylcholine CRC serial dilutions were prepared at a 10X stock solution in assay buffer prior to addition to assay plates. Calcium mobilization was measured at 25 degrees C using a FLEXstation II (Molecular Devices, Sunnyvale, CA) according to the following protocol. Cells were preincubated with test compound (or vehicle) for 1.5 min prior to the addition of the agonist, acetylcholine. Cells were then stimulated for 50 sec with a one of eight concentrations of the Acetylcholine CRC. The signal amplitude was first normalized to baseline and then as a percentage of the maximal response to acetylcholine. EC50 values for the Acetylcholine CRC alone (i.e. plus Vehicle) and in the presence of a fixed high concentration (30 uM final concentration) of each test compound were determined using GraphPad Prism (4.0c), which fit curves using standard non-linear regression (variable slope).This compound shifted the EC50 of acetylcholine in the calcium flux assay by greater than 13 fold. The compound was assigned 'Outcome' as 'Active' and 'Score' as '100'.2416http://www.vanderbilt.edu/mlscn1133CHRM5 cholinergic receptor, muscarinic 5 [ Homo sapiens ]1Average_%_Veh_MaxCalculated Average for Avg. Max vehicle concentration1152SD_Average_%_Veh_MaxCalculated Std. Deviation for the Avg. Max vehicle concentration1153SEM_Average_%_Veh_MaxCalculated S.E.M. for the Avg. Max Vehicle Concentration1154Average_%_Veh_MinCalculated Average for the Avg. Min Vehicle Concentration1155SD_Average_%_Veh_MinCalculated Stand. Dev for Avg. Min Vehicle Concentration1156SEM_Average_%_Veh_MinCalculated S.E.M for the Avg. Min Vehicle Concentration1157Cmpd Conc1 Rep1Value for compound + Ach at 0.0000001 uM replicate 1151.0000000116861e-07518Cmpd Conc2 Rep1Value for compound + Ach at 0.000001 uM replicate 1159.99999997475243e-07519Cmpd Conc3 Rep1Value for compound + Ach at 0.00001 uM replicate 1159.99999974737875e-065110Cmpd Conc4 Rep1Value for compound + Ach at 0.0001 uM replicate 1159.99999974737875e-055111Cmpd Conc5 Rep1Value for compound + Ach at 0.001 uM replicate 1150.001000000047497455112Cmpd Conc6 Rep1Value for compound + Ach at 0.01 uM replicate 1150.009999999776482585113Cmpd Conc7 Rep1Value for compound + Ach at 0.1 uM replicate 1150.1000000014901165114Cmpd Conc8 Rep1Value for compound + Ach at 1.0 uM replicate 11515115EC50_uM_Rep1Calculated EC50 for replicate 11516Fold_Shift_Rep1Calculated Fold Shift for Replicate 1.125417Cmpd Conc1 Rep2Value for compound + Ach at 0.0000001 uM replicate 2151.0000000116861e-075218Cmpd Conc2 Rep2Value for compound + Ach at 0.000001 uM replicate 2159.99999997475243e-075219Cmpd Conc3 Rep2Value for compound + Ach at 0.00001 uM replicate 2159.99999974737875e-065220Cmpd Conc4 Rep2Value for compound + Ach at 0.0001 uM replicate 2159.99999974737875e-055221Cmpd Conc5 Rep2Value for compound + Ach at 0.001 uM replicate 2150.001000000047497455222Cmpd Conc6 Rep2Value for compound + Ach at 0.01 uM replicate 2150.009999999776482585223Cmpd Conc7 Rep2Value for compound + Ach at 0.1 uM replicate 2150.1000000014901165224Cmpd Conc8 Rep2Value for compound + Ach at 1 uM replicate 21515225EC50_uM_Rep2Calculated EC50 for replicate 21526Fold_Shift_Rep2Calculated Fold Shift for Replicate 2125427Cmpd Conc1 Rep3Value for compound + Ach at 0.0000001 uM replicate 3151.0000000116861e-075328Cmpd Conc2 Rep3Value for compound + Ach at 0.000001 uM replicate 3159.99999997475243e-075329Cmpd Conc3 Rep3Value for compound + Ach at 0.00001 uM replicate 3159.99999974737875e-065330Cmpd Conc4 Rep3Value for compound + Ach at 0.0001 uM replicate 3159.99999974737875e-055331Cmpd Conc5 Rep3Value for compound + Ach at 0.001 uM replicate 3150.001000000047497455332Cmpd Conc6 Rep3Value for compound + Ach at 0.01 uM replicate 3150.009999999776482585333Cmpd Conc7 Rep3Value for compound + Ach at 0.1 uM replicate 3150.1000000014901165334Cmpd Conc8 Rep3Value for compound + Ach at 1 uM replicate 31515335EC50_uM_Rep3Calculated EC50 for replicate 31536Fold_Shift_Rep3Calculated Fold Shift for replicate 3125437Average_EC50_uMCalculated Avg EC50 for the first 3 replicates1538SD_EC50_uMCalculated Stand. Dev of EC50 for the first 3 replicates1539SEM_EC50_uMCalculated SEM for EC50 for the first 3 replicates1540Average_Fold_ShiftCalculated Average Fold Shift for the first 3 replicates125441SD_Fold_ShiftCalculated Stand. Dev of Fold Shifts for the first 3 replicates125442SEM_Fold_ShiftCalculated S.E.M. of Fold shifts for the first 3 replicates12543muscarinic acetylcholine receptor M5 [Homo sapiens]203731061Homo sapienshumantaxon9606muscarinic acetylcholine receptor M5 [Homo sapiens]21CR Plot Labels 1ConcentrationResponse02CR Plot Labels 2ConcentrationResponse03CR Plot Labels 3ConcentrationResponse02